Our broad objective is to further characterize the MHC class I pathway of antigen processing. Elucidation of this pathway is important because it plays a critical role in host defense against viruses and cancers. Moreover, insights into underlying mechanisms should be useful for understanding how pathogens evade immune responses and in designing approaches to improve vaccines and immunotherapies. The goal of Aim 1 is to identify the proteolytic steps and the enzymes involved in MHC class I antigen processing. Antigenic peptides must be of a precise size to be presented. To generate these peptides we hypothesize that there are at least two distinct proteolytic steps. The first step involves the cleavage of antigens into large oligopeptides. The second step refines the peptide to the appropriate size. We further hypothesize that the proteasome is the major mechanism responsible for this step and often generates the correct C-terminus of presented peptides while subsequent trimming is mediated by aminopeptidases. Using both intact cells and cell free systems, we will test these hypotheses, determine the subcellular compartments where these steps occur and identify the responsible enzymes activities. Aim 2 seeks to test the hypothesis that the proteases involved in antigen processing will be influenced by flanking residues and consequently flanking sequences will affect whether or not peptides are presented and the efficiency of this process. The obvious importance of this hypothesis is that understanding such effects would aid in predicting epitopes and designing more potent antigens for vaccines as well as giving insight into the phenomenon of immunodominance. Our experimental approach will systematically mutate flanking residues and then examine the effects of these changes on proteolysis in cell free systems and on the efficiency of peptide generation in vivo. Aim 3 seeks to discover and define new steps in the class I presentation pathway using a genetic approach. We will select for antigen presenting cells that have mutations which result in low surface expression of class I (impairment in any key step in the class I pathway will result in this phenotype). We will then focus our analysis on clones that have novel defects in the class I pathway. We have preliminary data that demonstrates the feasibility of this approach. Distinct mutations or steps will be identifies by genetic complementation analysis. The nature of the defects will be characterized biochemically and in antigen presentation assays. An important feature of our approach is the use of a genetically tractable system that will allow cloning of the mutated genes or genes that suppress the mutant phenotype.